The crucial role of homeobox genes in craniofacial development is well established. Changes in Hox gene expression result in severe craniofacial defects, involving the branchial arches, the primordia for palate shelf formation. Recent evidence shows that Hox genes act through regulating cell condensation and growth m the skeleton, the craniofacial bones and the palate. Folate supplementation protects against craniofacial abnormalities, indicating a functional interaction with developmental pathways. Common to the action of Hox genes and folate are (i) their effects on cell proliferation, (ii) their involvement m Retinoic Acid-mediated teratogenesis and (iii) their actions on specific craniofacial structures. We here propose to investigate the functional relationship of defects induced by folate-deficiency to those mediated by Hox genes. Our central hypothesis is that folate and Hox genes mediate cell growth in craniofacial structures through common cellular and molecular mechanisms. This will be investigated using two animal models with craniofacial defects: mice with genetic modifications in folate metabolism, and mice with genetic manipulations in Hox genes. The specific aims of this study are: (1) To define the role of folate and Hox genes in craniofacial development by analyzing cell proliferation in BrdU incorporation and apoptosis assays. (2) To examine the expression of folate pathway genes m Hox mutants. This will be done by in situ hybridization on embryos from transgenic mice with conditional expression of Hoxd-4 and Hoxc-8 directed specifically to branchial arch 2. (3) To analyze Hox gene expression in folate pathway mutants. These studies will use Folbp1 knockout mice and will identify which folate-dependent defects are mediated by particular Hox genes in specific craniofacial regions. (4) To determine the functional relationship of folate and Hox genes in the morphogenesis of craniofacial defects. This will be accomplished genetically, by combining mutations in the folate pathway and those in Hox genes. This will allow us to ascertain whether folate acts through Hox genes or vice versa, and how both act m the pathogenesis of specific craniofacial defects. Our long-term goal is to integrate the control of gene expression by Hox transcription factors and metabolic regulation m the proper morphogenesis of craniofacial structures. Knowledge about the interactions of genes with environmental factors will be important for designing therapeutic strategies and preventive measures against craniofacial defects.